The long term goals of the present application are to determine the catalytic mechanisms, three-dimensional structures and physiological function of bacterial N-acetyltransferases. As a result of the intensive genome sequencing efforts of the last decade, and modern bioinformatics approaches to the identification of protein superfamilies, some 10,000 members of the GCN5-related N-acetyltransferase (GNAT) family have been identified. In bacteria, these include family members whose function is (1) the acetylation of aminoglycoside antibiotics, (2) the N-terminal acetylation of the ribosomal proteins S5, S18 and L12, and (3) unknown. The specific aims of the current application are organized to address these three classes of bacterial N-acetyltransferases. Of the thousands of encoded bacterial GNAT proteins, only three are known to acetylate proteins. They are encoded by the rimI, rimJ and rimL genes that are presumed to function in the alpha-N-acetylation of their cognate substrates; the ribosomal S5, S18 and L12 proteins, respectively. The state of acetylation of the latter protein is correlated with bacterial growth, suggesting that reversible enzymatic acetylation/deacetylation is important in controlling bacterial growth. Bacterial resistance to antibiotics is a clinically significant problem that threatens current paradigms of antibacterial chemotherapy. Aminoglycosides were one of the first classes of antibiotics used in the treatment of bacterial infections, and act by specifically inhibiting bacterial protein synthesis. Clinically, the vast majority of resistance is due to the expression of enzymes that modify the drug, including enzymes that phosphorylate, adenylate or acetylate aminoglycosides. Of these three activities, the expression of aminoglycoside N-acetyltransferases is most prevalent in clinical isolates. We will continue our examination of bacterial aminoglycoside N-acetyltransferases. Finally, in the genomes of the important human pathogens, Salmonella typhimurium and Myeobaeterium tuberculosis, there are 29 and 20, respectively, predicted GNAT family members, for which only 8 and 4, respectively, have putative, annotated functions, most of which include the functions discussed above. We will develop reagents and methods to define the physiological substrates for these enzymes.